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A Great Divide: Regulation of Mitotic Transitions

Research Summary

Rosella Visintin is interested in how cells inherit the correct number of chromosomes. Her laboratory uses the budding yeast Saccharomyces cerevisiae as a model system to elucidate the molecular mechanisms that drive and control chromosome segregation during mitosis.

My laboratory is interested in understanding the molecular mechanisms that control cell division, the process by which a cell generates two genetically identical daughter cells. Cells need to replicate their chromosomes and faithfully distribute each copy to the daughter cells. To ensure that each cell receives only one copy of each chromosome, cell cycle events need to be coordinated in time and space. If these mechanisms fail then genomic integrity is lost, which can lead to cell death or the acquisition of proliferation abnormalities. My group focuses on mitosis, the phase of the cell cycle when replicated genomes are separated and packaged into daughter nuclei. We study chromosome segregation to understand how errors made during this process contribute to the transformation of a healthy cell into a cancerous one.

Mitosis consists of a highly choreographed sequence of events that lead to dramatic cellular reorganization. Although it is a continuous process, cytological changes allow it to be arbitrarily divided into subphases including prophase, prometaphase, metaphase, anaphase, and telophase. Three major transitions take place during mitosis: (1) the G2-to-M transition, when entry into mitosis is controlled; (2) the metaphase-to-anaphase transition, when sister chromatid separation is triggered; and (3) the M-to-G1 transition, when cells reverse the processes that led to mitotic entry and reset the conditions for a new round of cell division. In higher and lower eukaryotes, transitions 2 and 3 define mitotic exit. They are the focus of our laboratory.

Metaphase-to-Anaphase Transition: Chromosome Segregation
To ensure that chromosomes are correctly transmitted during cell division, replicated chromosomes (sister chromatids) must first be separated and then segregated between the daughter cells. Sister chromatid segregation occurs during anaphase and is triggered by dissolution of the cohesin complexes that hold the sister chromatids together. Cohesin is cleaved by separase, whose activity is restrained by securin (Figure 1). Securin, in turn, is controlled by a surveillance mechanism, the spindle assembly checkpoint (SAC), a signaling pathway that delays sister chromatid separation until all sister chromatids have correctly attached to the microtubules of the mitotic spindle. When the SAC is satisfied, cells can proceed to anaphase. Progression through anaphase is mediated by mitotic spindle activities. A focus of my lab is to obtain a molecular understanding of the regulatory networks that control sister chromatid separation and spindle dynamics. We recently found a budding yeast mutant that cannot proceed through anaphase regardless of having degraded securin and cleaved cohesin. Elucidating the molecular defects characterizing this double mutant will allow us to define a novel pathway that is essential for sister chromatid segregation.

M-to-G1 Transition: Mitotic Exit
Mitotic exit initiates with the down-regulation of cyclin-dependent kinase (CDK) activity, a family of kinases whose activity controls cell cycle progression. Next, the phosphate groups that CDKs added to their targets to allow cells to enter mitosis must be removed so that the cells can exit mitosis. During my postdoctoral work in Angelika Amon's laboratory, I found that in budding yeast, the Cdc14 phosphatase is important for both CDK down-regulation and the reversal of mitosis-promoting phosphorylation events. I also showed that Cdc14 activity is controlled by changes in its subcellular localization. The phosphatase is sequestered in the nucleolus by its inhibitor Cfi1/Net1 for much of the cell cycle. At anaphase, two regulatory networks, the Cdc14 early anaphase release (FEAR) network and the mitotic exit network (MEN), sequentially release Cdc14 from Cfi1. This sequential activation of Cdc14 triggers, in a wave-like manner, the dephosphorylation of distinct populations of CDK substrates and thus mitotic events at different stages of anaphase. The FEAR network and the MEN coordinate mitotic exit with different cell cycle events (Figure 2).

When I started my laboratory, I continued to work in this area of research because critically important questions about the control of exit from mitosis remained unanswered. In particular, I wished to determine how Cdc14 is inactivated after completion of mitotic exit and how the interaction of Cdc14 and Cfi1 is regulated.

Cdc14 Inactivation After Completion of Mitotic Exit
Studies in my laboratory showed that Cdc14 plays an important role in its overall regulation. Cdc14 contributes to its own activation via a feedforward mechanism coupled to a positive feedback loop whereby Cdc14, released by the FEAR network and the MEN, further activates the MEN. We showed that, once fully activated by the MEN, Cdc14 activates the anaphase promoting complex (APC/C) bound to its specificity factor Cdh1. This process not only rapidly inactivates CDKs and promotes exit from mitosis, it also initiates the return of Cdc14 into the nucleolus, where it binds to its inhibitor. By activating the APC/CCdh1, Cdc14 triggers the degradation of the Polo kinase Cdc5, a key factor in its own activation as a member of the FEAR network and activator of the MEN.

Understanding How the Cdc14-–Cfi1 Interaction is Regulated
Evidence has suggested that phosphorylation events mediate the release of Cdc14 from its inhibitor. Three kinases were implicated in the process: the polo-like kinase Cdc5, the mitotic CDK, and the most downstream MEN kinase Dbf2. Work in my laboratory showed that Cdc14 release requires the power of two kinases: Cdc5 and either Clb-CDK or the MEN. Our analysis of Cdc14 localization in MEN mutants overproducing Clb-CDKs also led to the finding that Cdc14 was not continuously released from the nucleolus but showed consecutive cycles of release and sequestration. Theoretical studies propose that oscillations are triggered by negative feedback loops. As described above, Cdc14 functions in a negative feedback loop to promote its own inactivation comprising APC/CCdh1, Cdc14, and the polo-like kinase Cdc5. We could show that this negative feedback loop underlies the oscillatory behavior of Cdc14 in the mutant. Intriguingly, oscillatory behaviors have been described for bud formation in yeast and centrosome duplication. A common theme emerges from these studies. Although these events are intrinsically capable of oscillating, they are limited to one occurrence by their coupling with the cyclin-CDK cell cycle engine.

Although we have established a framework of how mitotic exit is controlled in budding yeast, important questions remain to be addressed. For instance, what is the molecular mechanism that regulates the Cdc14–Cfi1 interaction? What is the significance of the requirement for two kinases underlying Cdc14 release? How is Cdc14 activation orchestrated and coordinated with different cell cycle events? Other questions regarding the pathways regulating Cdc14 activity are also unresolved. Why does the FEAR network consist of at least two branches? Which signals are sensed by the FEAR network? How does the FEAR network promote Cdc14 activation? We wish to find answers to these questions.

Research grants from the Italian Association for Cancer Research (AIRC) and the Giovanni Armenise–Harvard foundation provided partial support for these projects.

As of January 17, 2012